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SOP for Washing of HPLC Column After Use

Standard operating procedure to wash the HPLC column after analysis.


       To clean the column thoroughly after its each use to avoid any interference on repeated usage.

2.0  SCOPE

       This procedure is applicable for different groups of H.P.L.C columns used in Quality Control laboratory.


2.1  Doing      : Technical Assistant
2.2  Checking : Executive /Manager


       Head of the Department


5.1  For C18, C8, C6, Phenyl, (CN, Amine, Silica column.:)

5.1.1  Wash the column with the solvent which is use in individual product mobile phase preparation. eg. suppose water is use in mobile phase preparation then wash the column with water.
5.1.2  Allow the minimum 30 ml washing medium to flow through the column.
5.1.3  Disconnect the column from the HPLC unit and store in an allocated place.

5.2  For Protein pack column:

5.2.1  Wash the column with minimum 30 ml of 2.5 M acetic acid.
5.2.4  Disconnect the column from the H.P.L.C unit and store in an allocated place.

5.3  For L17 column :

5.3.1  Change the mobile phase to a mixture of 0.01 N H2SO: ACN : 85:15  v/v and flush at flow rate of 0.6 ml/min. minimum for 2 hrs.
5.3.2  Take care that flow rate should not exceed 0.6 ml/minute.
5.3.3  Flush the column with water at flow rate of 0.6 ml/min, for 2 hours.
5.3.4  Disconnect the column from the H.P.L.C unit and store in an allocated place.


6.1  H.P.L.C. = High Performance Liquid Chromatography
6.2  PSI =  Pound per square inch
6.3  ACN = Acetonitrile
6.4  H2SO4 = Sulphuric acid
6.5  nm = Nanometer
6.6  ml =  Millilitre
6.7  v/v = Volume by Volume

Related: Relative Response Factor in HPLC

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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